Advances in genetic recombination technology have allowed a variety of protein formulations to be offered in consistent amounts of supply. In order to stabilize, those formulations are provided either as a form where freeze-dried powder of protein component is packaged separately from a liquid diluent for dissolving it upon using, or as a protein solution formulation having an additive added to improve its stability.
Such protein containing formulations in some cases contain nonionic surfactant of polysorbates with a view to preventing protein adsorption on the container and stabilizing. For example, a granulocyte colony stimulating factor (G-CSF) formulation containing polysorbates as a stabilizer has been disclosed (JP 63-146826 A).
Polysorbates are polyoxyethylene sorbitan fatty acid esters and, depending on the species of the fatty acid ester, are designated 20 (monolaurate), 40 (monopalmitate), 60 (monostearate) and 80 (monooleate), each bonding to a polymer comprising about 20 moles of ethylene oxide units. Taking polysorbate 20 as an example, it can be produced by the following method:
Briefly, sorbitol is subjected to intramolecular dehydration to make sorbitan (2,6-type and 1,4-type), which in turn is reacted with lauric acid in the presence of NaOH as a catalyst to make a fatty acid ester, and the fatty acid esters then treated with ethylene oxide. In polysorbate 20, 1 mole of sorbitan monolaurate is bound to about 20 moles of ethylene oxide, and the molecule does not have a single structure. Polysorbates 20 and 80 are used most preferably in formulations containing proteins such as erythropoietin (EPO) and granulocyte colony stimulating factor (G-CSF).
Polysorbates are generally added to protein formulations-in very small-amounts of 0.001-3%, and in order to control the quality of the protein formulations, it is necessary to establish methods of verifying and quantifying such small amounts of polysorbates. Polysorbate 80 currently used in EPO formulations are not highly effective in preventing adsorption if its concentration is too low; on the other hand, if its concentration is too high, it promotes the decomposition of the protein. It is therefore required that the amounts of polysorbates in protein formulations be measured accurately.
The following methods may be employed to quantify polysorbates.    1) High performance liquid chromatography (HPLC): An alkyl ether of poly(ethylene oxide) is fluorescently labeled with 1-anthroylnitrile and thereafter analyzed on a reverse-phase column (M. Kudoh et al., J. Chromatogr., 287:337, 1984); in other methods, detection at a low wavelength of 220 nm is utilized with direct analysis on a reversephase column (RP18) (N. Garti et al., J. Am. Oil Chem. Soc., 60:1151, 1983) or a normal phase column (N. Garti et al., J. Liq. Chromatogr., 4:1173, 1981).    2) Gas chromatography (GC): A fatty acid produced by acid hydrolysis is converted to a methyl ester which is then analyzed (C. N. Wang et al., J. Am. Oil Chem. Soc., 61:581, 1983); alternatively, hydrobromic acid is allowed to act on the sample and the generated 1,2-dibromoethane is analyzed (Hisashi Tanaka et al., Suishitsu Odaku Kenkyu, 7:294, 1984).    3) Wickbold's method: After foam concentration, a Dragendorff reagent is added to form a precipitate, which is then dissolved and subjected to a potentiometric titration using pyrrolidine dithiocarbamate (R. Wickbold, Tenside Deterg., 9:173, 1972, etc.).    4) Frameless atomic absorption spectrometry: Cobalt in a combined complex of polysorbate and cobalt thiocyanate-ammonium is analyzed by atomic absorption spectrometry (A. Adachi et al., Eisei Kagaku, 29:123, 1983).    5) Colorimetry: A combined complex formed of the ethylene oxide portion of polysorbate and cobalt thiocyanate-ammonium is measured by absorbance (at maximum wavelength) (R. A. Greff et al., J. Am. Oil Chem. Soc., 42:180, 1965, etc.); other methods include a similar use of a complex with iron(III) thiocyanate (Shoji Murai: Bunseki Kagaku, 33: T18, 1984), as well as a method in which potassium ions are coordinated to polysorbate and an ion pair consisting of the potassium ion and the picrate ion is extracted with dichloroethane and subjected to colorimetry (L. Favretto et al., Intern. J. Environ. Anal. Chem., 14:201, 1983).
Among the methods mentioned above, the colorimetry using the cobalt thiocyanate complex has high sensitivity, is easy to perform, does not require any special equipment or reagents, and features the widest scope of applicability. In addition, this method is described as an identification test for polysorbate 80 in Japanese Pharmacopoeia (A Compendium of the 12th rev. Japanese Pharmacopoeia, B-639 (polysorbate 20) and D-891 (polysorbate 80), Hirokawa Shoten) and as an identification test for polysorbate 20 in British Pharmacopoenia (British Pharmacopoeia 1993, Vol.1, 525, London Her Majesty's Stationary Office) . Hence, the colorimetric approach using the cobalt thiocyanate complex is considered to be most suited as a method of quantifying polysorbates in drugs. Briefly, the method comprises adding an ammonium thiocyanate/cobalt nitrate reagent to an aqueous solution of the sample, further adding an organic solvent such as chloroform or dichloromethane to the solution, mixing the ingredients by shaking to transfer the cobalt thiocyanate complex to the organic solvent layer, and performing colorimetry of the cobalt thiocyanate complex.
However, if a sample solution containing a protein is tested by the same method, a protein aggregate may sometimes form between the water layer and the organic solvent layer, and lower the extraction rate of the polysorbate, eventually making it impossible to achieve accurate quantitation.
It is therefore required to develop a method by which the contents of polysorbates in protein containing solution samples can be determined in a simple and accurate manner.